JP3164814B2 - Preparation of homogenized cDNA library - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an equalized cDNA library (equalized cDNA library).
NA library) and its manufacturing method. More specifically, the present invention has been widely applied to research fields such as medicine and biology, such as cloning of unknown genes, identification of transcription regions on genome sequences or cosmid clones, and simultaneous identification of expression patterns of a large number of genes. The present invention relates to a useful homogenized cDNA library and a method for producing the same.

(Prior Art) In recent years, scientific elucidation of the tissues and mechanisms constituting living organisms and the accompanying technological applications have rapidly advanced, and the basics of genetic engineering have been established one after another.

Under these circumstances, various genes having structures or functions unique to species and individual tissues have been identified for genes of all living organisms from prokaryotes to eukaryotes including humans. While a great deal of knowledge has been accumulated, on the other hand, storing the individual genes thus obtained in a state where they can be used at any time as needed, is an additional requirement for that gene. It is a very important issue in terms of analysis and its medical, biological, and engineering applications.

Conventionally, when a specific gene is stored, a method of partially digesting the gene DNA, cloning each fragment into a cloning vector or the like, and preparing a so-called gene library from a collection of individual clones has been used. Have been. One of such gene libraries is a genomic library. In this method, all genes (genomic DNA) present in a particular organism are fragmented and individually cloned.

It is estimated that less than 1% of the genome of a particular organism encodes a protein, that is, the part that plays an important role in actually constructing an organism. That is, most of the information in the library is often unnecessary. A cDNA library clones only the part encoding this protein. This is based on mRNA present in cells, regardless of prokaryotes or eukaryotes, and using complementary transcriptase as a template
A (complementary DNA: cDNA) is synthesized, and this is individually cloned to prepare a gene library.

This library can be used in a variety of ways not found in genomic libraries, such as expression of its protein in animal cells by connecting it to an expression vector. Have been.

(Problems to be Solved by the Invention) However, in the case of a cDNA library, different from a genomic library, different types can be produced depending on the cells from which the material is obtained. For most multicellular organisms, individual cells are specialized,
In other words, it is organized as an aggregate of various different types of cells. Moreover, each cell has a set of genes, but the gene group to be expressed is determined by the type of cell, and only the expressed genes are
This is because it is transcribed into mRNA.

Further, one cell has many types of expressed genes. For example, in the case of hepatocytes, the number of types of expressed genes is estimated to reach 20,000 to 30,000. Moreover, each of them does not exist in a uniform ratio. In the case of hepatocytes, about 16% of the amount of mRNA is occupied by one kind of mRNA encoding a protein called albumin. In addition, all transcribed cells are stimulated by estrogen in chicken oviduct cells.
50% of the mRNA amount is one type of mRNA called ovalbumin. On the other hand, the gene with the lowest content is only a fraction of the total mRNA.
It is estimated to be less than 0.0005%, a difference of 10
It reaches ten thousand times.

Therefore, in the conventional method, that is, when cDNA is synthesized using mRNA transcribed from an expressed gene as a template and cloned individually to prepare a cDNA library, the difference in the content of each mRNA species is the difference. This is directly reflected in the difference in the content of each type of clone constituting the library. That is, in the case of a hepatocyte cDNA library,
Approximately 16% of the total are cDNA clones of the same species, while the few will be only 0.0005%.

For this reason, a library containing a cDNA species having a low content rate becomes large. For example, in the liver, the total of all types of mRNA is at most tens of thousands, but in a conventional library, unless the size is several tens to several millions, it does not include all.

When a cDNA library is used, one cDNA is actually required for each type of gene, and the cDNAs present in the library are not only redundant, but also required for searching for the desired cDNA. It also hinders. Of course, even in a conventional cDNA library containing a large number of such extra cDNA clones, when targeting a specific gene whose base sequence is known, for example, by a known method such as a hybridization probing method, The cDNA of interest can be identified and isolated relatively easily. Also,
Even when the nucleotide sequence is unknown, a screening method has been established when an antibody against the protein exists.

The problem is in identifying and isolating the cDNA encoding the protein using the function as an indicator. In this case, each cDNA must be inserted into an expression vector, the protein must be produced in the cell, and the effect of the protein on the cell must be examined. In addition, there are a large number of genes that cannot be identified or isolated unless a huge number of clones in the library are searched one by one. recent years,
The use of cDNA libraries to search for such unknown genes is becoming important.
The presence of a large number of redundant extra cDNAs in the library is a major problem.

For the same reason, the types of all the genes that can be expressed are limited (it is said to be about 100,000 in humans), but even one set of the cDNA species has not been prepared.

If such a thing could be made, it would no longer be necessary for each researcher to make a cDNA library in each case.

The present invention has been made in view of the above circumstances, and overcomes the problems of the conventional cDNA library, and provides a homogenized cDNA library containing each mRNA, that is, the molecular species of the expressed gene at a more equal ratio. The purpose of the present invention is to provide a method for preparing a rally and such a homogenized cDNA library.

(Means for Solving the Problems) The present invention provides an mRNA for solving the above problems.
After denaturing the double-stranded cDNA synthesized using the as a template and re-associating, the cDNA that has re-associated with the double strand and the single-stranded cDNA are separated, and this single-stranded cDNA is converted into the double-stranded cDNA. Homogenized cDNA characterized by being synthesized and cloned
A method for preparing a library and a homogenized cDNA library prepared by such a method are provided.

In a preferred embodiment of the present invention, the single-stranded cDNA is fragmented by arranging a cohesive end at its 3 'end in advance, and the cDNA to be cloned or its fragment is amplified by PCR. I have.

 Hereinafter, the configuration of the present invention will be described in detail.

First, in the present invention, a cDNA library of the gene can be prepared for any kind of cell of any kind.

And, in principle, one of the genes that can be expressed for each species
The production of a cDNA library containing the entire set will also be included in the project.

Extraction and purification of mRNA serving as a template for cDNA can be performed according to a known conventional method. For example, the extraction and purification can be performed as follows, for example.

That is, for example, since eukaryotic cytoplasmic mRNA is concentrated in polysomes that are complexes with ribosomes, polysomes are first separated by fractional centrifugation or centrifugation in a sucrose solution or its density gradient. Next, mRNA is extracted from the polysome using a mixed solution of phenol and chloroform, or phenol or hot SDS-phenol saturated with a pH 9 buffer. The mRNA thus extracted is further purified into a specific mRNA depending on the size by using sucrose density gradient centrifugation or gel electrophoresis.

When synthesizing cDNA from the mRNA obtained in this way,
Known methods can be used. For example, many mRNAs present in the cytoplasm of eukaryotic cells have 10
This poly A sequence has a poly A sequence consisting of 0 to 200 bases.
Then, for example, an oligo (dT) primer is annealed, and a reverse transcriptase is used as a primer to synthesize a polynucleotide comprising a DNA complementary to the RNA strand. Further, the polynucleotide is subjected to an alkali treatment to decompose the RNA strand into a single-stranded cDNA. In the single-stranded cDNA, a hairpin loop is formed at the 3 ′ end, and this is used as a primer to complement the hairpin loop. The cDNA strand is synthesized into a double-stranded cDNA.

In the case of preparing a normal cDNA library, this double-stranded cDNA is treated at both ends with, for example, S1 nuclease, etc., and further attached with cohesive ends, and then cloned using an appropriate cloning vector. In the present invention, instead of cloning all of the obtained cDNA,
They are selected and homogenized to equalize each cDNA species.

In order to perform such homogenization, first, all of the obtained double-stranded cDNA is denatured (denatura
to dissociate into single strands, and further react the single-stranded cDNA in a reassociation buffer at an arbitrary temperature (for example, 65 ° C.) to reassociate into double-stranded cDNA again.
Let it. At this time, each single-stranded cDNA naturally re-associates with each other.However, the more overlapping cDNAs present in the reaction solution, the faster the re-association, and the lower the content of the cDNA, the lower the re-association. Takes time to complete. Therefore, if the reassociation reaction is stopped at an appropriate time, the double-stranded cDNA and the single-stranded cDNA are present in the reaction solution. Of these, the double-stranded cDNA group is mostly occupied by cDNA species that originally existed in large numbers, so the remaining single-stranded cDNA group had a content of unsuccessful reassociation in time. There will be a single strand of high cDNA species and a single strand of low cDNA content that is inherently less likely to reassociate. Therefore, the double-stranded cDNA and single-stranded cDNA present in the reaction solution are separated by a known method, for example, using a hydroxyapatite column, and only the single-stranded cDNA is double-stranded again as described above. In this group of cDNAs, the difference in the content of each cDNA species is greatly reduced. Furthermore, by repeating such denaturation and reassociation treatment a plurality of times, a set of cDNAs containing each cDNA species at an equal ratio can be finally obtained.
A homogenized cDNA library can be prepared by inserting and ligating each NA to a cloning vector.

The denaturation and reassociation treatments used to measure such homogenization may be performed, for example, in the case of selecting mRNA specifically expressed in only one cell from mRNA of two types of cells, or in any case. Whose expression is specifically induced by the above-mentioned means (for example, the action of a specific hormone)
Is widely used, for example, when selecting from mRNAs that are unique to the cell. For example, when preparing a cDNA library of only the mRNA species specifically expressed in the cell (A) for the cell (A) and the cell (B),
Generally, the mRNA of the cell (A) is converted into single-stranded cDNA, and the mRNA of the cell (B) is associated with the single-stranded cDNA.
The procedure for cloning NA is performed, and at that time, the mR expressed in both cells (A) and (B) is commonly used.
In order to completely remove the NA species, it is necessary to add a large excess of the mRNA of the cell (B) to the cDNA of the cell (A). However, in the present invention, since it is essential that all types of cDNAs do not fall off at all and remain at a uniform ratio, the cDNAs to be subjected to the reassociation reaction are each complementary strands.
It is an absolute condition that the ratio be exactly one to one.
This also theoretically ensures that all mRNA species (cDNA species) are included in the homogenized library without exception.

However, when a homogenized cDNA library is prepared by the above method, there is a risk that the following two problems may occur depending on the type of target cells and the like.

One is the shedding of required cDNA species during denaturation and reassociation treatments for homogenization. Usually, when a single-stranded cDNA is subjected to an associating reaction, ones whose sequences are complementary associate with each other to form a double-stranded cDNA. But actually, it ’s different
Even if the single strands of cDNA derived from mRNA are 50
If it has a homology region of about bp, those single-chain cD
NA may form a double strand. For example,
There are about 10 types of mRNAs that encode a protein called actin, but since their protein coding regions are very homologous, when single-stranded cDNAs of each of these are associated, they encode actin in the same way, However, cDNAs of different species may form double strands. As mentioned above,
In the present invention, as a result of the reassociation reaction within a certain time,
Double-stranded cDNA is excluded from the library. Therefore, when single-stranded cDNAs of different species are re-associated with each other as in the case of the above-mentioned actin gene, an incomplete library in which several types of required cDNAs have been dropped will result. In order to overcome such a problem, according to the present invention, a full-length cDNA synthesized from mRNA is fragmented into several hundred base pairs by mechanical shearing force in advance, and only the cDNA fragment in the 3 'terminal region is cloned. Also provided is a method for preparing a modified cDNA library.

That is, generally, a region having high homology with another gene on one mRNA is limited to only a small part such as a protein coding region, and conversely, the untranslated region on the 3′-terminal side has the specificity. Is known to be high. Therefore, this 3 'terminal region
In the cDNA, single-stranded cDNAs of different species do not form double strands even by the reassociation reaction, and there is no danger that the required cDNA species will be dropped from the library.

In this method, when the cDNA fragment of the 3 'terminal region is selectively cloned, for example, the full-length cDNA
Before fragmenting NA, it is sufficient that only the fragment can be ligated to the cloning vector by attaching a sticky end to its 3 'end.

Next, another problem in the production method of the present invention is as follows.
As a result of removing and homogenizing most of the redundant cDNA species present in large numbers, the amount of cDNA to be finally cloned is reduced, and the efficiency of integration of the cDNA into the cloning vector is reduced. . However, such a problem can be solved by amplifying the cDNA to be cloned using a PCR method (Polymerase Chain Reaction) or the like. For example, if the single-stranded cDNA remaining after the reassociation reaction is amplified by the PCR method, a sufficient amount of cDNA to ensure good cloning efficiency can be easily obtained.

Therefore, in the examples described below, Ltk ^- cells, which are mouse fibroblast cell lines,
The example which produced the library is shown.

Example 1 (Purification of mRNA and Synthesis of cDNA) mRNA of Ltk ^- cell was extracted and purified, and synthesized into cDNA.

First, Ltk ^- cells were cultured in a medium containing 10% fetal bovine serum, and total RNA was isolated from cells in the growing phase according to a conventional method. Next, mRNA having a poly A sequence was extracted from these RNAs using an mRNA purification kit (Pharmacia),
Purified.

Furthermore, the mRNA of Ltk ^- cell thus prepared is
Using the mRNAs of the neo gene (1.3 kb) and the tk gene (1.8 kb), which are foreign genes, as control markers, one each
0 w / w% and 0.0005 w / w% were added and used as a template for cDNA.

5 μg of the thus obtained mRNA was ligated to an oligo (dT) -Not I primer (5′-AATTCGCGGCCCGCT
CDNA was synthesized using TTTTTTTTTTTTTT-3 ′ (Promega).

Next, the synthesized full-length double-stranded cDNA was transferred to the Branson Sonifier.
Using 250 (Branson) to fragment to 200-400 bp
After sorting by agarose gel electrophoresis, the ends were treated with T4 DNA polymerase.

Example 2 (Amplification of cDNA fragment by PCR method) The double-stranded cDNA fragment obtained in Example 1 was amplified by PCR method.

Since each end of the cDNA fragment obtained in Example 1 is a brand end, the following linker primer (LL-RI)
Was attached.

Since the protruding ends of the LL-RI do not adhere to each other, a single molecule of the linker specifically binds to both ends of the cDNA fragment.

After attaching LL-RI to both ends of the cDNA in this manner,
Using LL-RIA oligomers shown in Fig. 1 as primers
PCR was performed to amplify the cDNA fragment. That is, 1 ng of cD
NA was added to the 100 μl reaction, followed by a 25 minute cycle at 94 ° C. for 2 minutes, 50 ° C. for 2 minutes, and 72 ° C. for 2 minutes.
Repeated times, the cDNA was amplified.

Next, the amplified cDNA was extracted with an equal amount of phenol-chloroform-isoamyl alcohol saturated with TE,
Further, free primers not incorporated into the cDNA chain were removed using Centricon-100 (Amicon).

Example 3 (Uniformization of cDNA) Denaturation and reassociation treatment were repeated three times on the cDNA fragment amplified in Example 2, and the content of each cDNA species was equalized.

That is, in the first homogenization (EI) and the second homogenization (E II), 20 μg of the amplified cDNA was added to 1.5 ml Eppe.
Dissolve in 10 μl of distilled water in an ndorf tube, and add an equal volume of 2 × hibridization solution (0.24 M NaH ₂ PO ₄ [pH 6.8],
1.64 M NaCl, 2 mM EDTA, 0.2% SDS) was added. Also,
In the third homogenization (E III), 1
00 μg of cDNA was added.

In order to prevent the evaporation of each of these sample solutions, the surface was covered with light oil and boiled for 5 minutes to denature the cDNA to form a single strand. Next, EI and EII were subjected to a reassociation reaction at a temperature of 65 ° C. for 12 hours, and EIII was subjected to a reassociation reaction at the same temperature for 24 hours.

As an indicator of the degree to which complementary DNA strands reassociate, C
There is an ot value, but the Cot value when making EI, E II and E III (mol.l
iter ^-1 .sec) were 130,130,1100, respectively.

As a result of such denaturation and reassociation, a double-stranded cDNA and a single-stranded cDNA were obtained, which were filled with hydroxyapatite according to a conventional method.
Separation in a column at a temperature of ° C., desalting of single-stranded cDNA fragments with Centricon-100, amplification of these by the same PCR method as in Example 2, and re-singulation into double-stranded Was synthesized.

Example 4 (Preparation of cDNA library and evaluation of homogenization thereof) A cDNA fragment subjected to three times of homogenization treatment in Example 3 was cloned, and cDNA was obtained from a colony of the transformant.
A library was made.

First, the double-stranded cDNA obtained in Example 3 was digested with restriction enzymes EcoR I and Not I, and this was cut into a plasmid vector pBluesc
It was ligated into the EcoR I-Not I cleavage site of ript SK (-). Further, this vector was introduced into a host cell, and the host cell was transformed to form a colony.

Next, the homogeneity of the cDNA library thus obtained was evaluated by the following method. That is, as an index indicating the degree of homogenization of the library, an overlap index (Abun
dance variation). This is the cDN
It is a numerical value obtained by dividing the content% of the clone that exists most redundantly among the clones constituting the A library by the content% of the least clone. In practice, it is physically impossible to check the content of each clone for all clones.
Note that there are two methods for obtaining the overlap index. One is a method of performing colony hybridization using a specific cDNA as a probe according to a conventional method, and measuring the ratio of positive colonies in the library. In this case, it is preferable to use as many types of probe cDNA as possible. The second is a method of randomly picking up cDNA clones from a library, determining their nucleotide sequences, and comparing the individual nucleotide sequences to estimate how many identical clones exist.

Here, using the former method, cD prepared as described above
The duplication index of the NA library was calculated. Probes used were neo, tk, and Ltk ^- cell expressed genes mouse β-actin, el added as control markers.
F-4A, Vimentin, IAP, EF-1α, and ATPase-6 genes in total of eight types.

The results are shown in Table 1. Table 1 shows the number of positive colonies for each probe for each of the cDNA library (S) according to the conventional method and the homogenized cDNA library (EI, EII, EIII) of the present invention, which were shown as comparative examples. The content percentage is shown in parentheses.

As is clear from Table 1, the cDNA library (S) obtained by the conventional method has more than 1600 (estimated about 2
(000) The duplication index was reduced to 40 in the library of the present invention (EIII) which had been subjected to three homogenization treatments.
The theoretically completely homogenized cDNA library refers to a library having an overlapping index of 1, but if experimental errors and the like are taken into account, the ratio of the largest content to the smallest content is calculated. A factor of 40 seems to be a sufficient achievement for the degree of homogenization.

(Effect of the Invention) As described in detail above, according to the present invention, cloning and identification of unknown genes, simultaneous identification of expression patterns of a large number of genes, and one set of all genes that can be expressed in organisms including humans The present invention provides a homogenized cDNA library which is widely useful in medical biology research fields and genetic engineering fields, such as preparation of a cDNA library having the same, and a method for preparing the same.

Continuation of front page (56) References Nucleic Acids Res. , 18 (1990 Aug. 25), (16), p. 4833-1842 Science, 222 (1983), (4620), p. 135-139 Nucleic Acids Res. , 18 (1990 Oct. 25), (19), p. 5705-5171 Protein Enzyme Nucleic Acid Extra Issue Genome Analysis Research, February 10, 1993, Kyoritsu Shimbun, Vol. 38, No. 3, p. 420-428 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00 C12Q 1/68 BIOSIS (DIALOG) JICST file (JOIS) WPI (DIALOG)

Claims (4)

(57) [Claims] (1) denaturing a double-stranded cDNA synthesized using the entire mRNA expressed in a cell A as a template, re-associating the cDNA within an arbitrary time, and then combining with the cDNA re-associated with the double strand; Separating the unstranded cDNA, synthesizing the single-stranded cDNA into a double-stranded cDNA and cloning each cDNA, the cDNA containing the entire mRNA expressed in the cell A and containing the most abundant cDNA species CDNA with the difference in the content of the least cDNA species of 100 times or less
A method for preparing a homogenized cDNA library, which comprises preparing a library. 2. A double-stranded cDNA synthesized using mRNA as a template,
2. The method for preparing a homogenized cDNA library according to claim 1, wherein the cohesive end is previously arranged at the 3 'end to fragment the fragment. 3. The cDNA or a fragment thereof to be cloned is subjected to PCR.
The method for producing a homogenized cDNA library according to claim 1 or 2, wherein the cDNA library is amplified by a method. 4. A homogenized cDNA library prepared by the method according to any one of claims (1) to (3),
Includes cDNA of total mRNA expressed in
A cDNA library, wherein the difference between the species and the content of the least cDNA species is 100 times or less.